Lab. Investigation Of Mp

Laboratory investigations of Malarial Parasite

Dr. Md Moyez Uddin Director Institute of Public Health Sujan Chandra Mondal

Tests for Malarial Parasites Microscopic test 1.Peripheral blood film study 2.Quantitive Buffy Coat (QBC) test

Non microscopic test Rapid Diagnostic Tests (RDTs) Para Sight F test OptiMal Assay The immunochromatographic test (ICT Malaria P. f. test) Polymerase Chain Reaction Detection of antibodies by Radio immuno assay, immunofluorescence or enzyme immuno assay

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Procedure of Diagnosis ♦ Collection of blood ♦Preparation ♦Stain

-Giemsa stain (1:20 dilute) 30min

-Field’s stain 4-5 second ‘A’ Polychrome methyline blue ‘B’ Eosine

-Leishman stain Sujan Chandra Mondal

Preparation of PBF Step 2

Step 1

Hold the third finger of the left hand and wipe its tip with spirit/Savlon swab; allow to dry

Prick the finger with disposable needle/lancet; allow the blood to ooze out

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Preparation of PBF Step 3

Step 4

Take a clean glass slide. Take another clean Take 3 drops of blood 1 slide with smooth edges cm from the edge of the and use it as a slide, take another drop spreader... of blood one cm from the first drop of blood Sujan Chandra Mondal

Preparation of PBF Step 5 Step 6

and make thick and thin smears. Allow it to dry

Prepared smear. (Slide number can be marked on the thin smear with a lead pencil.)

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Thick film of MP ♦ Microscopic

view RBC disappeared

Streaks of blue cytoplasm with detached nuclear dot appearance like ‘coma’ or ‘exclamation mark’ WBC remain unchange

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Thick film  Advantage

♦ Parasite density

-Number of parasites counted in each microscopic field -density considered in thick film 20 or more /field : high density 2-19 / field : medium density 1 or less ; low density -It is important to report parasite density

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Rapid detection of parasite (*) Concentrating parasite 20 fold than thin film Guide to Rx for testing efficacy of antimalarial drug

 Disadvantage - species cannot detected ( *) 1 microscopic field of thick film equavalent to 50 microscopic field of thin film

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Thin film ♦ Certain rules before

declared negative -upper & lower margin of the tail end of film should examine thoroughly because parasite mostly numerous here -minimum 100 field must examined -time taken for examintion at least 8 to 10 min.

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Diagnostic points:Red Cells are not enlarged. Rings appear fine and delicate and there may be several in one cell. Some rings may have two chromatin dots. Presence of marginal or accole forms. It is unusual to see developing forms in peripheral blood films. Gametocytes have a characteristic crescent shape appearance. However, they do not usually appear in the blood for the first four weeks of infection. Maurer's dots may be present.

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Diagnostic points:Red cells containing parasites are usually enlarged. Schuffner's dots are frequently present in the red cells as shown above. The mature ring forms tend to be large and coarse. Developing forms are frequently present.

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Diagnostic points :Ring forms may have a squarish appearance. Band forms are a characteristic of this species. Mature schizonts may have a typical daisy head appearance with up to ten merozoites. Red cells are not enlarged. Chromatin dot may be onSujan theChandra inner Mondal surface of the ring.

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Diagnostic points :Red cells enlarged. Comet forms common (top right) Rings large and coarse. Schuffner's dots, when present, may be prominent. Mature schizonts similar to those of P. malariae but larger and more coarse

P. vivex

P.falciparum

P.malariae

P.ovale

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QBC test

♦new method

♦involves staining of the centrifuged and

compressed red cell layer with acridine orange and its examination under UV light source. ♦key feature-centrifugation & concentration of

the red blood cells in predictable area of the QBC tube, making detection easy and fast. Parasitized RBC less dense than normal ones & concentrate just below the leukocytes, at the top of the erythrocyte column. The float forcs all the surrounding red cells into the 40 micron space between its outside circumference and the inside of the tube. Since the parasites contain DNA which takes up the acridine orange stain, they appear as bright specks of light among the non-fluorescing red cells. Virtually all of the parasites found in the 60 microliter of blood can be visualized by rotating the tube under the microscope. Sujan Chandra Mondal

Fluorescense UV ray microscopy

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Other Rapid test (RDT’s) of MP ♦ new method

OptiMal Assey Kit

OptiMal Assay result Sujan Chandra Mondal

Immunochromatographic (ICT) Tests for Malaria Antigens ( HRP-2, LDH, Aldolase) ♦ based on the capture of the

parasite antigens from the peripheral blood using either monoclonal or polyclonal antibodies against the parasite antigen targets.

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can target the histidine-rich protein 2 of P. falciparum, a pan-malarial Plasmodium aldolase, and the parasite specific lactate dehydrogenase do not require a laboratory, electricity, or any special equipment.

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Comparison between peripheral smear and QBC test for detecting malaria Peripheral smear

QBC

Method

Cumbersome

Easy

Time

Longer, 60 - 120 minutes

Faster, 15 - 30 minutes

Sensitivity

5 parasites/µl in thick film and 200 / µl in thin film

Claimed to be more sensitive, at least as good as a thick film

Specificity

Gold standard

? False positives, artifacts may be reported as positive by not-so-well-trained technicians

Species identificatio n

Accurate, gold standard

Difficult to impossible

Cost

Inexpensive

Costly equipment and consumables

Acceptabilit y

100%

Not so

Availability

Everywhere

Limited Sujan Chandra Mondal

Peripheral Smear

Rapid Diagnostic Tests

Format

Slides with blood smear

Test strip

Equipment

Microscope

Kit only

Training

Trained microscopist

Test duration

20-60 minutes or more

Test result

Direct visualization of the parasites

Color changes on antibody lines

Capability

Detects and differentiates all plasmodia at different stages

Detects malaria antigens (P PMA/pLDH) from asexual sexual forms of the par

Detection threshold

5-10 parasites/µL of blood

1 00-500/µL for P. falciparum for non-falciparum

Species differentiation

Possible

Cannot differentiate amon falciparum species; mixed in of P. falciparum and non-fa appear as P. falciparu

Quantification

Possible

Not possible

Differentiation between sexual and asexual stages

Possible

Not possible

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'Anyone with a little trai 5-30 minutes

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